Rapamycin glucoside type compound as well as enzyme method preparation and application thereof
A technology of rapamycin and glucoside, which is applied in the preparation, application, and drug combination of sugar derivatives, and can solve problems such as difficult selective protection, many by-products, and not being environmentally friendly
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Embodiment 1
[0041] Example 1 Expression of glycosyltransferase BsGT-1, in vitro glycosylation reaction, and preparation of rapamycin glucoside
[0042] The inventor screened and identified a glycosyltransferase BsGT-1 (CUB50191) capable of efficiently glycosylating rapamycin, and its protein sequence has been published.
[0043] The recombinant plasmid containing pET28a-BsGT-1 was transformed into Escherichia coli BL21 (DE3), and the resulting transformant was transferred to 2ml of LB medium supplemented with kanamycin (final concentration was 40 μg / ml), 200r / min, at 37 Cultivate at ℃ for 12 hours, then transfer 2ml of the seed solution to 100ml of LB medium (the final concentration of kanamycin is 40μg / ml), and expand the culture at 37℃. When the OD value reaches about 0.6-0.8, add Isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, and then transferred to a shaker at 16° C., 200 r / min, cultivated for 24 hours, centrifuged at 8000 r / min for 5 min, and collect...
Embodiment 2
[0048] Example 2 Compound 1, Compound 2 and Compound 3 Structure Identification
[0049] The structures of compound 1, compound 2 and compound 3 were determined by high-resolution mass spectrometry and nuclear magnetic resonance analysis, according to figure 2 and image 3 , UHPLC-ESI-Q-TOF high-resolution mass spectrometry gives compound 1 and compound 2 quasi-molecular ion peaks [M+Na] + They are m / z 1098.5886 and 1098.5913 respectively, thus confirming that 1 and 2 are monoglucosides of rapamycin. Simultaneously, the monosaccharide can also be obtained from the H NMR spectra of compounds 1 and 2 ( 1 H NMR, Figure 5 , Figure 10 ) and carbon spectrum ( 13 C NMR, Figure 6 , Figure 11 ) to be confirmed. Two-dimensional NMR spectroscopy determined the link between the sugars of compounds 1 and 2 and the main chain of rapamycin ( Figure 7-9 , Figure 12-14 ).
[0050] In the HMBC spectrum of compound 1, H-40 is related to C-1' and H-1' is related to C-40, thus co...
Embodiment 3
[0052] Example 3 Antifungal activity test of rapamycin glucoside
[0053] Screening method: half-dilution method
[0054] Cell lines: Candida albicans; Fusarium graminearum and Pythium aphanidermatum
[0055] Action time: 48 hours
[0056] experimental method:
[0057] 1. Use YPD liquid medium (dissolve 10g yeast extract, 20g peptone in 900ml water, high pressure 115°C, 15min, add 100ml 20g glucose) to cultivate Candida albicans, when OD 600 When it reaches 0.3, carry out dilution coating and dilute to 10 5 -10 6 The concentration of cells / ml was used for the antifungal test. The initial drug concentration was set at 32 μg / ml, and half-diluted in a 96-well plate. The inhibition rates of the corresponding concentrations of compounds 1, 2, and 3 were as follows:
[0058] The inhibitory rates of compound 1 to Candida albicans were 96.08% (32 μg / ml), 95.20% (16 μg / ml), 94.75% (8 μg / ml), 94.26% (4 μg / ml), 94.26% (2 μg / ml) , 94.01% (1 μg / ml), 92.83% (0.5 μg / ml), 88.98% (0.25 μ...
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